The evolution of the obligate human pathogen Neisseria gonorrhoeae has been shaped by selective pressures from diverse host niche environments and antibiotics. The varying prevalence of antibiotic resistance across N. gonorrhoeae lineages suggests that underlying metabolic differences may influence the likelihood of acquisition of specific resistance mutations. We hypothesized that the requirement for supplemental CO 2 , present in approximately half of isolates, reflects one such example of metabolic variation. Here, using a genome-wide association study and experimental investigations, we show that CO 2 dependence is attributable to a single substitution in a β-carbonic anhydrase, CanB. CanB 19E is necessary and sufficient for growth in the absence of CO 2 , and the hypomorphic CanB 19G variant confers CO 2 dependence. Furthermore, ciprofloxacin resistance is correlated with CanB 19G in clinical isolates, and the presence of CanB 19G increases the likelihood of acquisition of ciprofloxacin resistance. Together, our results suggest that metabolic variation has affected the acquisition of fluoroquinolone resistance., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)